Hello! I am struggling with getting the diatoms from the rock in order to identify them. I have followed the common procedures (a mix of equal amounts of HCl and H2O2 - 30%) but the rock is not dissolving. Also, I have lowered the concentration, but still no result.
I even tried to freeze and unfreeze the sample in distilled water but no success.
I have tried to keep them for 40 seconds in the sonic bath, but unfortunately that breaks the frustules (Fig. 2).
I know for sure that I have diatoms in the rock. Also, the cement is siliceous, which probably the HCl and the peroxide is not working.
Is there an other way of identifying the fossil diatoms? Its pretty hard to identify them on the rock (Fig 1).
Thank you,
Emilia
Emilia:
You may try this method and do let me know if you succeed:
Glueckauf!
Best
Syed
The tough problem is the siliceous cement of the rock, because anything used to chemically break it up (e.g. hydrofluoric acid) would also affect and damage the valves and frustules...
Dear Emilia,
I would like to recommend you firstly to pulverize your siliceous rock and than just used a cold diatom preparation method, it means other chemicals and without heating the chemical soup. This cold preparation method is much more friendly for small frustules. Maybe it will help you. This cold method up-grated and modified by me for a very special cases, for a very complicated samples with a lot of small particles, debris, siliceous dust, it is following:
Diatom preparation, Method II (after Bohuslav Uher):
The material and chemicals: sample, tubes, pipettes, distilled water, HCl, H2SO4, HNO3
The first day - homogenize sample and replace hydrochloric acid (HCl) (time: about 1 hour)
1. Start at 9:00 am with thawed samples, document everything in the lab book!
2. Number clean tubes (e.g., No. 1, 2, etc.) and pipettes (# 1, 2, ...) and prepare in a rack,
3. Mix well the water in the thawed sample with numbered pipette (homogenize)
4. Pour at least 3 mL of the sample into the tube with the pipette immediately from the well-homogenized sample
5. Centrifuge the tubes for 10 minutes (3000 rpm - revolutions per minute;
RCF = 0.86525 m.s-2 - Relative Centrifugal Force)
6. Mix well with distilled water, wash and centrifuge for 10 minutes (3000 rpm)
7. Then slowly replace the remaining water with 20-30 mL HCl hydrochloric acid: Caution, it can foam very intensively! (at the beginning, just test with a few drops how thick it foams)
8. Mix well / homogenize the sediment with HCl acid with a pipette
9. Let it work until the next day
The second day - exchange acids instead of HCl .... H2SO4-HNO3 mixture (time: about 2 hours)
10. At 9:00 the next day, centrifuge the sample for 10 minutes, replace the HCl acid from the sample with distilled water, and wash three times (centrifuge for 10 minutes), mixing well, taking about 10-20 minutes between each wash
11. Replace the residual water with approx. 10:30 o'clock with 20-30 mL H2SO4-HNO3 mixture (always use the protective mask with acid filter!)
12. Thoroughly homogenize the sediment with the pipette
13. Let it work until the next day
The third day - washing and dripping (time: about 5 hours)
14. Centrifuge the tubes the next day at 09:00 (always use the mask with acid filter!)
15. Replace the H2SO4-HNO3 mixture with distilled water
16. Wash four to six times, mix well, centrifuge for 10 min (3000 rpm), pause for about 10-20 minutes between each wash
17. Repaint part of the sediment in a new tube
18. Wash the part of the sediment in the new tube (mark with letter A for 1, B for 2 ... etc ...) three times with distilled water, homogenize well, take a break of about 10-20 minutes between each wash
19. Prepare two parallel slides per sample, i. four coverslips
20. Drizzle small suspension into the new tube and four different concentrations of the sample on four coverslips (all slides label A01, A02, B01, B02 ... etc).
21. Allow to air dry for one day at the air temperature
The fourth day - Naphrax and control of the preparations (time: about 4 hours)
22. Embed with the Naphrax
23. Place a sufficiently large drop of NAPHRAX on the cooled, dry coverslips, which are placed on the ceramic plate and covered with a whitish layer. The correct amount of containment must be determined by experiment.
24. It should be spread as far as possible over the coverslip without flowing over its edge.
25. Set the adjustable heating plate to 250 °C
26. Heat for about 30 seconds to about 120 °C until the containment medium has spread as free of bubbles as possible to the cover glass edge.
27. A template in slide size with marked position of the cover glass is helpful.
28. On a piece of cardboard or wood, the preparations are allowed to cool
29. Autoevaluation - Self-control of quality - Microscopic analysis
30. At least 1000x magnification to make quality control
31. The controlled properties of diatomaceous preparations:
32. Determination and enumeration Plausible (abbr. "BZ") = at least 500 diatom shells per coverslip, samples with very low diatom abundance must then have more than 300 shells per three coverslips
33. Number of cups (abbr. "SM") = at 1000x magnification of 7 to 15 trays per field of view
34. Thickness under cover glass (abbr. "D") = diatom shells should be in one layer at most, no 3D effects should not be found
35. Fine structure (abbreviated "FS") = detailed diatom structure must be clearly visible (small species of Achnanthes, Navicula, Caloneis, etc.)
36. Mineral Veil (abbr. "MS") - Categories: ok / little / none, moderate, much, very much
37. Mineral particles (abbr. "MP") - Categories: ok / little / none, moderate, much, very much
38. Distribution (abbr .: "V") - Diatom shells should not form large islands and aggregates
39. Comment (abbr. "Kom") - when it comes to extreme wetland biotopes (heavily boggy, cloudy waters, stagnant trenches), e.g. In the preparation of the diatomaceous suspension at least has to be carried out six to eight washes with distilled water. Partly due to inorganic streaking and strong mineral turbidity, the suspension had to be additionally washed several times alternately with ethanol and distilled water.
Good luck, hopefully it could help you.
Sincerely
Bohuslav
Dear all,
Thank you kindly for your reply!
@Syed - I have tried something similar and unfortunately is not working. I used saturated sulphuric acid and potassium permanganate in equal amounts. It`s reacting with the rock because I have high organic matter content, but its not breaking the rock. So I get a lot of fizz but nothing in suspension.
@Marco - I also end up with the last solution the hydrofluoric acid, but as you mention I can destroy the diatoms frustules very easily.
@Bohuslav - the rock is not reacting with the HCl since I have a very little carbonate content (below 2%), and I cannot mill the sample because I will destroy the diatoms.
Since the rock has the siliceous cement will be quite hard to remove it from the diatoms without breaking them.
Thank you all!
Cheers,
Emilia
Emilia:
Give a try to the same method as for extracting Radiolaria from the Chert as shown in this link:
Best
Syed
https://books.google.co.in/books?id=qc88AAAAIAAJ&pg=PA579&lpg=PA579&dq=Extracting+Diatoms+from+siliceous+cement&source=bl&ots=E_T62uUHEu&sig=uXHhGD8Q3x3WODs44S5ckszWBD8&hl=en&sa=X&ved=2ahUKEwixrb6un73cAhWObn0KHQRnBsQQ6AEwCHoECAgQAQ#v=onepage&q=Extracting%20Diatoms%20from%20siliceous%20cement&f=false
Dear Syed,
Thanks a lot for your reply! Unfortunately, I cannot used hydrofluoric acid in the lab due to safety reasons.
All the best,
Emilia
Indeed it is very dangerous because the preparation of siliceous materials develops silicon tetrafluoride whih is neurotoxic... Moreover, it would destroy or heavily damage your diatom valves...
Stay safe & good luck.
Marco
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